1. Field of the Invention
The present invention relates to a hand-held chemical vapor detector for detecting biological substances in an indoor and outdoors setting. More specifically, the present invention relates to a plasma chromatograph (PC) vapor detector that is interfaced to a biological sample processing and transfer introduction system. The biological sample processing was accomplished by quartz tube thermal decomposition (TD), and the resultant vapor was transferred by gas chromatography (GC) to the PC detector. This system is comprised of a thermal decomposition module, gas chromatography module and a plasma chromatograph detector. These components are connected in a series fashion. The device is referred to as a Biological Classifier System (BCS). The BCS can be described as a hyphenated device where two analytical dimensions (the GC and PC), in series, allow the separation and isolation of individual components from the thermal decomposition of biological analytes.
2. Brief Description of Related Art
Recent and current events around the world have highlighted the possibilities for deliberate outdoor dissemination of harmful biological substances, and at least 12 countries are known to have some degree of biological warfare program capabilities. Alleged biological terrorism attacks in Japan and threats on U.S. domestic commercial establishments have increased significantly in the past five years. Reports of alleged localized aerosol releases and hoax domestic biological terrorism in the form of postal mail packages, allegedly with spores of the pathogenic Bacillus anthracis and Yersinia pestis (bubonic plague) organisms, serve to exacerbate the problem.
Desirable goals in effectively countering the biological warfare and terrorism applications of harmful biological agents include their ready detection and possible identification in a relatively short period of time. The detection of biological aerosols, particularly that of bacterial cells and spores, is an important component of U.S. military biological programs. A portion of these programs consists of analytical instrumentation to effect trigger, detection, and identification responses for the presence of bacterial aerosols.
Analytical investigations of aerosols have relied on a diverse set of approaches over the last three decades from experimental determinations of generated aerosols from bulk solutions to real time analyses of ambient outdoor particles. These investigations have included inorganic (salts) and organic particulates as well as bioaerosols that include microorganisms, fungi, and pollen. An accounting of the most prevalent techniques and instrumental methods appears constructive with respect to the present analytical detection method of biological aerosols.
Traditional methods for the characterization of aerosols consist of sampling ambient air and collecting/concentrating them on various matrices (1-3). These biological aerosol particulates are then subjected to sample detection techniques such as polymerase chain reaction (PCR) (1,2,4,5), colony plate count or most probable number (MPN) (1,4,6,7), bioluminescence from inherent adenosine triphosphate (ATP) (4,8), phase-contrast microscopy (4), and immunoassay (1,4,9). The bacterial aerosol samples were characterized by these traditional detection techniques in either an off-line or on-line fashion.
Pseudomonas fluorescens bacteria were aerosolized and directed to an agar plate with a laser sizing system placed after the aerosol generator. This was an important development in that a relation could be produced between the total number of particles and the number of bacterial-colony-forming units on the agar growth plate (10).
Mass spectrometric methods have had a long and rich history as analytical vehicles for investigating compositional properties of artificial and outdoor man-made aerosols as well as ambient organic and inorganic aerosols under off-line or on-line analysis conditions.
Laser microprobe mass analysis (LAMMA) has been used in an off-line fashion to investigate aerosol particles. Particles were collected or placed on a matrix or wire mesh and were introduced into a vacuum. A microscope guides a laser beam to a selected particle or spot on the matrix surface where ions desorb and are analyzed by a time-of-flight mass spectrometer (TOFMS). Relatively low molecular weight species were usually observed, and known species mostly represented the inorganic salt fraction of samples such as Mycobacterium leprae (11,12), B. anthracis. B. thuringiensis, and B. cereus (13,14) as well as ionized species of particulates including polyaromatic hydrocarbons (PAH) (15) and salt species. Two recent review articles on LAMMA document the principal and extensive applications of TOF and Fourier-transform mass-spectrometer analyzers in the analysis of laser generated ions of single aerosol particles (17,18) that were placed or impacted on matrix supports.
The TOFMS field evolved to where an aerosolized suspension of biological particulates could be introduced into a vacuum as single particles. This procedure, particle analysis by mass spectrometry (PAMS), used either a hot rhenium filament to pyrolyze individual bacterial particles (19-22) or a laser to desorb and ionize species from particles (22,23). These biological particles, including Pseudomonas putida, Bacillus cereus, and Bacillus subtilis var. niger were generated from an ethanol-water suspension. Linear quadrupole mass spectral determinations mainly produced unknown pyrolysis fragments and inorganic salt-derived species.
A similar system, developed by Gieray, Reilly, Yang, Whitten, and Ramsey (24) used an ion trap mass spectrometer detector. From a bulk water suspension, bacterial aerosol particles were sensed and a trigger was provided by the particles passing through two argon ion laser beams. An excimer laser ablated the particle in the ion trap so as to produce ions. An improvement on this basic design was that of a TOF system (25-27) replacing the quadrupole mass spectrometer designs. This allowed for faster mass spectral scanning of ions from particles generated from bulk suspensions or directly from laboratory ambient air. Salt particles as well as tobacco smoke and soot were analyzed.
Hars et al. used a combined electrodynamic balance/ion trap mass spectrometry technique for trapping and stabilizing aerosolized particles of polystyrene and Bacillus subtilis spores, followed by laser fragmentation/ionization to obtain mass spectra of the ions generated during a 450-mJ pulse from a Nd-YAG laser. They demonstrated the feasibility to use this technique for chemical and physical characterization of single cells of microorganisms and other components of respirable aerosols (28,29). In other work, an aerosol particle-sizing laser was interfaced to a laser thermal desorption/ionization beam for TOFMS analysis on organic and inorganic compounds (30,31).
Prather et al. published a series of evolving articles with the concept of size, aerodynamic diameter, chemical composition and composition class, and temporal characterization of outdoor aerosol particles (32-39). The centerpiece was a transportable aerosol concentrator, dual time-of-flight mass spectrometer. Positive ions are analyzed in one tube and negative ions, from the same particle, are analyzed in the second time-of-flight tube. As an example, over a period of four days, pyrotechnic explosives (fireworks) particles were monitored in the atmosphere: monitoring sites were 0.5 and 3 miles from the explosion sources (37). Further examples of this technology are the characterization of automobile emissions (36) where metals, oxides, hydroxides, and polyaromatic hydrocarbons (PAH) were detected, and in the temporal monitoring of the nitric acid to hydrochloric acid heterogeneous chemistry that occurs in atmospheric aerosols over the ocean-land mass interface (39).
Gas chromatography (GC)-MS has been used for the trace analysis of bacteria and fungi in organic dust aerosols from environments such as hospitals and homes (40-44) and biotechnology processes (45,46). The air was continually sampled for bacteria for a period of 24 h, and the bacteria on the filter were processed for the extraction of specific biochemical compounds for GC-MS analysis.
Biological aerosol analyses using analytical instrumentation were mainly relegated to controlled investigations in laboratory settings as related earlier. Only a few investigations can be found in the literature concerning the real-time detection of bacterial aerosols in outdoor scenarios, and the detection methods were primarily spectroscopic in nature. Real-time detection of Bacillus spore aerosols from the dissemination of bulk suspensions in outdoor testing areas was accomplished by a light detection and ranging (LIDAR) system. This is a remote detection system where a 1064-nm laser beam was used to interrogate a Bacillus subtilis var. niger aerosol plume approximately perpendicular to the beam (6,47). The backscattered radiation was collected by receiver telescope optics and used in the determination of bacterial aerosol presence.
The fluorescence of an aerosol of B. subtilis from bulk suspension was detected as single particles with a fluorescence particle-counter instrument in outdoors and under indoor, controlled conditions. An argon laser beam of 488 nm interrogated a beam of particles by monitoring emission in the 530 to 550-nm range (48,49). Flavin compounds in the bacteria were surmised, because the bacterial component responsible for the emission at 530 to 550 nm and non-biological interferences displayed no fluorescence activity in the emission bandwidth. Another report dealt with combining the 488-nm argon laser, which produces size-scattering and visible fluorescence, with a 266-nm pulsed laser to produce 300-500-nm UV-VIS fluorescence of the proteinaceous bacterial components (50) from the generated aerosols. Similar experiments with B. subtilis were shown with a 325-nm helium-cadmium laser excitation source with fluorescence monitoring at 420 to 580 nm (51).
The first TD-GC-PC analyses of biological materials, including Bacillus spores and nucleic acids, appear to have been reported by Meuzelaar, Kim, Arnold, Kalousek and Snyder (52). This was followed by systematic TD-GC-PC studies of various biopolymers relevant to bioagent detection (53) as well as potential interferences, culminating in a recent PhD thesis by Thornton (54).
Prototype portable GC-PC concept systems were shown to successfully separate headspace vapors from complex liquid mixtures of analytes (55-57).
Laboratory testing of the prototype of the currently fielded TD-GC-PC version was performed under controlled sample introduction of bacterial suspensions (58). Moreover, a preliminary presentation of a spore aerosol investigation in the field with the prototype TD-GC-PC was reported recently (59). Finally, a more comprehensive study of the detection of outdoor aerosols of Bacillus subtilis var. niger (BG) bacterial spores, Gram-negative Erwinia herbicola (EH) bacteria and ovalbumin protein with TD-GC-PC has recently been performed (60-62).
Technologies are very different from TD-GC-PC and can produce better or less information. Almost all have not been tested in the field and some are large and are a logistics burden.
The present investigation provides for the determination of desirable properties of generated biological aerosols.
A biological classification system that is made of a thermal decomposition tube for processing a biological sample and producing a resultant vapor, a gas chromatography module interfaced with the thermal decomposition tube by a three-way injection valve, the gas chromatography module for receiving the resultant vapor from the thermal decomposition tube, and a plasma chromatograph vapor detector interfaced via a GC/PC interface with the gas chromatography module for receiving resultant vapor from the gas chromatography module. In the biological classification system of the present invention, the thermal decomposition tube, the gas chromatography module and the plasma chromatograph vapor detector are connected in series for separation, isolation and classification of individual components from the thermal decomposition of biological analytes introduced into the thermal decomposition tube.